JPWO2020122049A1 - Animal cells, methods for producing animal cells and methods for producing target proteins - Google Patents

Abstract

An object of the present invention is to provide an animal cell capable of producing a target protein with high productivity, a method for producing the animal cell, and a method for producing the target protein using the animal cell. According to the present invention, an animal cell having a gene encoding a target protein and a foreign gene encoding SNAT2 and linked to a promoter and overexpressing SNAT2; the animal cell. ; And a method for producing a target protein using the above animal cells is provided.

Description

The present invention relates to animal cells expressing a protein of interest. The present invention relates to the above-mentioned method for producing an animal cell and the above-mentioned method for producing a target protein using the animal cell.

In the production of biopharmaceuticals such as antibodies, fed-batch culture is often used to improve the state of cells by adding nutrients to the culture medium in order to improve the productivity of antibodies. Further, in the production of biopharmacy such as antibody, in order to improve the productivity of antibody, the culture solution is continuously filtered and discharged, while a fresh medium containing a nutritional component is continuously supplied to the culture tank. , Perfusion culture method is often used.

In Patent Document 1, as a method capable of producing a protein in a high production amount, a cell in which alanine aminotransferase is strongly expressed and a DNA encoding a desired polypeptide is introduced is cultured, and the desired polypeptide is obtained. A method for producing a polypeptide, including producing it, is described.

International Publication WO 2009/020144

In Fedbatch culture, long-term cell culture is possible with a higher cell density than when no additional nutrients are added, but the cell viability rate in the latter half of the culture is affected by the accumulation of waste products secreted from the cells. There is a problem that the product cannot be increased by extending the culture period.
In addition, in order to cultivate cells at high density in the perfusion culture method, it is necessary to collect and supply 1 to 3 times the culture volume per day for the purpose of supplying nutrients and discharging waste products from the system. Since it is necessary, the problem is that the culture cost is high.

An object to be solved by the present invention is to provide an animal cell capable of producing a target protein with high productivity. Another object of the present invention is to provide a method for producing an animal cell and a method for producing a target protein using the animal cell.

As a result of diligent studies to solve the above problems, the present inventor affects the amount of protein production by forcibly expressing the SNAT2 (Sodium-dependent neutral amino acid transporter-2) gene (gene name is SLC38A2) in CHO cells. As a result, improvement of antibody productivity (Qp) and improvement of antibody production amount could be achieved. The present invention has been completed based on the above findings.

That is, according to the present invention, the following invention is provided.
<1> An animal cell having a gene encoding a target protein and a foreign gene encoding SNAT2 and linked to a promoter, and overexpressing SNAT2.
<2> The animal cell according to <1>, wherein overexpression is constitutive.
<3> The animal cell according to <1> or <2>, wherein the foreign gene encoding SNAT2 has a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 1.
<4> The animal cell according to any one of <1> to <3>, wherein the foreign gene encoding SNAT2 contains the nucleotide sequence shown in SEQ ID NO: 1.
<5> The animal cell according to any one of <1> to <4>, wherein the animal cell is a CHO cell.
<6> Any of <1> to <5>, which comprises a step of introducing a gene encoding a target protein and a foreign gene encoding SNAT2 and linked to a promoter into an animal cell. The method for producing an animal cell according to Kaichi.
<7> The method according to <6>, wherein the step of introducing a foreign gene encoding SNAT2 and linked to a promoter is performed by electroporation.
<8> A method for producing a target protein, which comprises culturing the animal cells according to any one of <1> to <5>.
<9> The method according to <8>, wherein the culture is fed-batch culture.
<10> The method according to <9>, wherein the seeded cell density of the cell culture is 0.2 × 10 ⁶ cells / mL or more and 5 × 10 ^{6 cells / mL or less.}
<11> The method according to <10>, wherein the viable cell rate during the culture period is 60% or more during the entire period.
<12> The method according to <8>, wherein the culture is a perfusion culture.
<13> The method according to <12>, wherein the seeded cell density of the cell culture is 0.2 × 10 ⁶ cells / mL or more and 1 × 10 ^{7 cells / mL or less.}
<14> The method according to <13>, wherein the viable cell rate during the culture period is 90% or more during the entire period.

According to the animal cell of the present invention, the target protein can be produced with high productivity.

FIG. 1 shows the results of measuring the antibody concentration in the culture supernatant of cells after gene transfer. FIG. 2 shows the results of measuring antibody productivity (Qp) per cell in the culture supernatant of cells after gene transfer. FIG. 3 shows the results of measuring the cell size after gene transfer. FIG. 4 shows the results of measuring the cumulative number of viable cells during the culture period.

Hereinafter, embodiments for carrying out the present invention will be described in detail.
The numerical range indicated by using "~" in the present specification means a range including the numerical values before and after "~" as the minimum value and the maximum value, respectively.

[Animal cells]
The animal cell of the present invention is an animal cell having a gene encoding a target protein and a foreign gene encoding SNAT2 and linked to a promoter, and the above-mentioned SNAT2 is overexpressed.

The expression of the SNAT2 gene activates the mTORC1 / S6K pathway in the mouse liver (Nature Communications 6, Article number: 7940 (2015)), suggesting a relationship between the expression of the SNAT2 gene and the expression of the mTOR gene. It has been reported that cell proliferation is suppressed and cell size is increased by activation of mTOR in rat cells (Genes Dev. 2002 Jun 15; 16 (12): 1472-1487). It has also been shown that the larger the cell size, the higher the production of protein products (Cytotechnology 34: 59-70, 2000). However, the relationship between SNAT2 gene expression and recombinant protein production is unclear.

In Patent Document 1, the decrease in survival rate was moderated due to the effect of introducing alanine aminotransferase, but since the period of 60% or less is long, there is concern about the influence on antibody quality. Further, since the antibody productivity (Qp) per cell is lowered by the introduction of the taurine transporter, there is a problem that even if the cell density is increased, it does not lead to the production amount. On the other hand, in the present invention, antibody productivity (Qp) per cell is improved by enhancing the expression of the SNAT2 gene involved in protein biosynthesis.

<Target protein>
In the present invention, the type of the target protein is not particularly limited, and for example, a recombinant polypeptide chain, a recombinant secretory polypeptide chain, an antigen-binding protein, a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, a bispecific antibody, and an Fc fusion. Proteins, fragmented immunoimmunoglobulins, single-stranded antibodies (scFv). The protein of interest is preferably a human antibody, a humanized antibody, a chimeric antibody, or a mouse antibody. Examples of the fragmented immunoglobulin include Fab, F (ab') ₂ , Fv and the like. The class of antibody is not particularly limited, and any class such as IgG such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, etc. may be used, but IgG and IgM are preferable when used as a medicine.

Human antibodies include all antibodies having one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all variable and constant domains are derived from human immunoglobulin sequences (fully human antibodies).
Humanized antibodies are less likely to elicit an immune response when administered to human subjects and / or induce a severe immune response compared to non-human species antibodies. Has a sequence different from that of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions so that In one embodiment, the heavy and / or light chain framework and certain amino acids within the constant domain of a non-human antibody are mutated to produce a humanized antibody. In another embodiment, the constant domain from a human antibody is fused to the variable domain of a non-human species.

A chimeric antibody is an antibody in which different variable regions and constant regions derived from each other are linked. For example, an antibody consisting of a variable region of a heavy chain and a light chain of a mouse antibody and a constant region of a heavy chain and a light chain of a human antibody is a mouse-human heterologous chimeric antibody. A recombinant vector expressing a chimeric antibody can be prepared by ligating a DNA encoding a variable region of a mouse antibody with a DNA encoding a constant region of a human antibody and incorporating this into an expression vector. By culturing the recombinant cells transformed with the above vector and expressing the integrated DNA, the chimeric antibody produced during the culture can be obtained.

Bispecific antibodies are antibodies made by chemical methods or cell fusion that recognize two different antigen specificities. A method for producing a bispecific antibody is to combine two immunoglobulin molecules with a cross-linking agent such as N-succinimidyl 3- (2-pyridyldithiol) propionate or S-acetylmercaptosuccinic acid anhydrate. Methods, methods of binding Fab fragments of immunoglobulin molecules to each other, and the like have been reported.

The Fc fusion protein refers to a protein having an Fc region and includes an antibody.
Fab _is a monovalent fragment having the V _L, V _{H, C L} and _{C H} 1 domain.
F (ab') ₂ is a divalent fragment having two Fab fragments linked by a disulfide bridge in the hinge region.
The Fv fragment has the single-armed _VL and _VH domains of the antibody.
A single chain antibody (scFv) is an antibody in which the _VL and _VH regions are joined via a linker (eg, a synthetic sequence of amino acid residues) to form a continuous protein chain, wherein the linker is: It is long enough to fold the protein chain over itself to form a monovalent antigen binding site.

The gene encoding the protein of interest can be obtained by a method known to those skilled in the art. When the target protein is an antibody, DNA encoding the L chain and DNA encoding the H chain of the antibody can be used.

The DNA encoding the L chain and the DNA encoding the H chain of the antibody can be prepared as follows. MRNA is extracted from hybridomas, cells, phages, ribosomes, etc. that have genes that express antibodies. CDNA is prepared from this mRNA by a reverse transcription reaction using reverse transcriptase. Each gene is obtained by amplifying the L-chain gene or H-chain gene by PCR using a primer having a complementary base sequence with the L-chain gene or H-chain gene and cDNA, and binding to a cloning plasmid.

The DNA encoding the L-chain fragment of the antibody and the DNA encoding the H-chain fragment can be prepared as follows. MRNA is extracted from hybridomas, cells, phages, ribosomes, etc. that have genes that express antibodies. CDNA is prepared from this mRNA by a reverse transcription reaction using reverse transcriptase. Each gene fragment is obtained by amplifying the L-chain gene fragment or H-chain gene fragment by PCR using a primer having a complementary base sequence with the L-chain gene fragment or H-chain gene fragment and cDNA, and binding to the cloning plasmid.

<SNAT2 and SNAT2 genes>
In the present invention, the origin of SNAT2 is not particularly limited, and a foreign gene encoding SNAT2 derived from mammals such as humans, monkeys, mice, rats, and hamsters can be used. In the present invention, the foreign gene refers to a gene introduced from the outside into an animal cell. Moreover, even if the endogenous gene is amplified and the gene is introduced, it is regarded as a foreign gene.

The nucleotide sequence and amino acid sequence of human SNAT2 are shown in SEQ ID NOs: 1 and 2 in the sequence listing.

Nucleotide sequence of human SNAT2 gene (SEQ ID NO: 1)
ATGAAGAAGGCCGAAATGGGACGATTCAGTATTTCCCCGGATGAAGACAGCAGCAGCTACAGTTCCAACA
GCGACTTCAACTACTCCTACCCCCACCAAGCAAGCTGCTCTGAAAAGCCATTATGCAGATGTAGATCCTGA
AAACCAGAACTTTTTACTTGAATCGAATTTGGGGAAGAAGAAGTATGAAACAGAATTTCATCCAGGTACT
ACTTCCTTTGGAATGTCAGTATTTAATCTGAGCAATGCGATTGTGGGCAGTGGAATCCTTGGGCTTTCTT
ATGCCATGGCTAATACTGGAATTGCTCTTTTTATAATTCTCTTGACATTTGTGTCAATATTTTCCCTGTA
TTCTGTTCATCTCCTTTTGAAGACTGCCAATGAAGGAGGGTCTTTATTATATGAACAATTGGGATATAAG
GCATTTGGATTAGTTGGAAAGCTTGCAGCATCTGGATCAATTACAATGCAGAACATTGGAGCTATGTCAA
GCTACCTCTTCATAGTGAAATATGAGTTGCCTTTGGTGATCCAGGCATTAACGAACATTGAAGATAAAAC
TGGATTGTGGTATCTGAACGGGAACTATTTGGTTCTGTTGGTGTCATTGGTGGTCATTCTTCCTTTGTCG
CTGTTTAGAAATTTAGGATATTTGGGATATACCAGTGGCCTTTCCTTGTTGTGTATGGTGTTCTTTCTGA
TTGTGGTCATTTGCAAGAAATTTCAGGTTCCGTGTCCTGGAAGCTGCTTTGATAATTAACGAAACAAT
AAACACCACCTTAACACAGCCAACAGCTCTTGTACCTGCTTTGTCACATAACGTGACTGAAAATGACTCT
TGCAGACCTCACTATTTTATTTTCAACTCACAGACTGTCTATGCTGTCCAATTCTGATCTTTTCATTTG
TCTGTCATCCTGCTGTTCTTCCCATCTATGAAGAACTGAAAGACCGCAGCCGTAGAAGAATGATGAATGT
GTCCAAGATTTCATTTTTTGCTATGTTTCTCATGTATCTGCTTGCCGCCCTCTTTGGATACCTAACATTT
TACGAACATGTTGAGTCAGAATTGCTTCATACCTACTCTTCTATCTTGGGAACTGATATTCTTCTTCTCA
TTGTCCGTCTGGCTGTGTTAATGGCTGACCCTGACAGTACCAGTAGTTATTTTCCCAATCCGGAGTTC
TGTAACTCACTTGTTGTGTGCATCAAAAGATTTCAGTTGGTGGCGTCATAGTCTCATTACAGTGTCTATC
TTGGCATTTACCAATTTACTTGTCATCTTTGTCCCAACTATTAGGGATATCTTTGGTTTTATTGGTGCAT
CTGCAGCTTCTATGTTGATTTTTATTCTTCCTTCTGCCTTCTATATCAAGTTGGTGAAGAAAGAACCTAT
GAAATCTGTACAAAAAGATTGGGGCTTTGTTCTTCCTGTTAAGTGGTGTACTGGTGATGACCGGAAGCATG
GCCTTGATTGTTTTGGATTGGGTACACAATGCACCTGGAGGTGGCCAT

Amino acid sequence of human SNAT2 gene (SEQ ID NO: 2)
MKKAEMGRFSISPDEDSSSYSSNSDFNYSYPTKQAALKSHYADVDPENQNFLLESNLGKKKYETEFHPGT
TSFGMSVFNLSNAIVGSGILGLSYAMANTGIALFIILLTFVSIFSLYSVHLLLKTANEGGSLLYEQLGYK
AFGLVGKLAASGSITMQNIGAMSSYLFIVKYELPLVIQALTNIEDKTGLWYLNGNYLVLLVSLVVILPLS
LFRNLGYLGYTSGLSLLCMVFFLIVVICKKFQVPCPVEAALIINETINTTLTQPTALVPALSHNVTENDS
CRPHYFIFNSQTVYAVPILIFSFVCHPAVLPIYEELKDRSRRRMMNVSKISFFAMFLMYLLAALFGYLTF
YEHVESELLHTYSSILGTDILLLIVRLAVLMAVTLTVPVVIFPIRSSVTHLLCASKDFSWWRHSLITVSI
LAFTNLLVIFVPTIRDIFGFIGASAASMLIFILPSAFYIKLVKKEPMKSVQKIGALFFLLSGVLVMTGSM
ALIVLDWVHNAPGGGH

As a foreign gene encoding SNAT2,
(1) A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(2) A gene encoding a protein having SNAT2 function, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, or (3) SEQ ID NO: 2. A gene encoding a protein having SNAT2 function, consisting of an amino acid sequence having 85% or more (more preferably 90% or more, particularly preferably 95% or more, most preferably 98% or more) sequence identity with the described amino acid sequence. ;
Can be used.

The SNAT2 function means a function of activating the mTORC1 / S6K pathway. It is known that in rat cells, cell proliferation is suppressed and cell size is increased by activation of mTOR.

As a foreign gene encoding SNAT2,
(4) A gene consisting of the nucleotide sequence shown in SEQ ID NO: 1;
(5) A gene encoding a protein having a SNAT2 function, which comprises a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1.
(6) A gene encoding a protein having a SNAT2 function, which comprises a base sequence that hybridizes under stringent conditions to the complementary sequence of the base sequence shown in SEQ ID NO: 1; or (7) Set forth in SEQ ID NO: 1. A gene encoding a protein having a base sequence having 90% or more (more preferably 95% or more, particularly preferably 98% or more) sequence identity with the base sequence of SNAT2 function:
Can also be used.

The "1 or several" in the "amino acid sequence in which one or several amino acids are deleted, substituted or added" is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. This means, particularly preferably 1-3.

Sequence identity in the present invention refers to a value calculated by the following formula.
% Sequence identity = [(same number of residues) / (longer alignment length)] x 100
Sequence identity in the two amino acid sequences can be determined by any method known to those of skill in the art, BLAST ((Basic Local Sequence Search Tool)) program (J. Mol. Biol. 215: 403-410, 1990). Etc. can be used to determine. The "longer alignment length" in the denominator means that the longer alignment length is used as the denominator when comparing the two alignments.

The "1 or several" in the "base sequence in which one or several bases are deleted, substituted or added" is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. This means, particularly preferably 1-3.

"Hybridizing under stringent conditions" means "hybridizing under moderate or high stringent conditions", which will be recognized by those skilled in the art. be able to. For moderate stringent conditions, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Vol. 1, 7.42-7.45 Cold Spring Harbor Laboratory Press, 2001. Moderate stringent conditions are, for example, a prewash solution of 5 × SSC, 0.5% SDS, 1.0 mmol / L EDTA (pH 8.0) in a nitrocellulose filter, about 50 at about 40-50 ° C. Hybridization conditions for% formamide, 2 × SSC to 6 × SSC (or other similar hybridization solution in about 50% formamide at about 42 ° C., such as Stark's solution), and about 60. Cleaning conditions of ° C., 0.5 × SSC, 0.1% SDS can be mentioned. Higher stringent conditions can also be readily determined by one of ordinary skill in the art, eg, hybridization and / or washing at higher temperatures and / or lower salt concentrations than the moderately stringent conditions described above. include. For example, hybridization conditions as described above and washing at 68 ° C., 0.2 × SSC, 0.1% SDS can be mentioned. Here, the composition of 1 × SSC is 150 mmol / L NaCl, 15 mmol / L sodium citrate, pH 7.4. SDS is sodium dodecyl sulfate and EDTA is ethylenediaminetetraacetic acid.

Information on the amino acid sequence and base sequence of non-human SNAT2 is shown below. The following numbers are NCBI (National Center for Biotechnology Information) Gene No. Is shown.
ID: 29642 rats (Rattus norvegicus)
ID: 67760 Mouse (Mus musculus)
ID: 566537 Zebrafish (Dario rerio)
ID: 338844 Boss taurus
ID: 417807 Chicken (Gallus gallus)
ID: 702253 Rhesus macaque (Maca mulatta)
ID: 1010833348 Cat (Felis catus)
ID: 10000525644 Pig (Sus scrofa)
ID: 101149564 Gorilla (Gorilla gorilla)

For non-human SNAT2, as in the case of human SNAT2,
(2A) A gene encoding a protein having SNAT2 function, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in a predetermined amino acid sequence;
(3A) A protein having an SNAT2 function, which comprises an amino acid sequence having 85% or more (more preferably 90% or more, particularly preferably 95% or more, most preferably 98% or more) sequence identity with a predetermined amino acid sequence. Gene to encode;
(5A) A gene encoding a protein having a SNAT2 function, consisting of a base sequence in which one or several bases are deleted, substituted or added in a predetermined base sequence;
(6A) A gene encoding a protein having SNAT2 function, which consists of a base sequence that hybridizes under stringent conditions to a complementary sequence of a predetermined base sequence; or (7A) 90% or more of a predetermined base sequence (7A) A gene encoding a protein having a base sequence having a sequence identity of 95% or more, particularly preferably 98% or more) and having a SNAT2 function:
May be used.

The foreign gene encoding SNAT2 is linked to the promoter.
The promoter is not particularly limited as long as it can function in host animal cells and express SNAT2. The promoters include CMV promoter (cytomegalovirus promoter), EF1α promoter (human polypeptide chain elongation factor gene promoter), SV40 promoter (siaminevirus 40 promoter), β-actin promoter, and MMLV-LTR promoter (Molony mouse leukemia). The long terminal repeat promoter of the virus), or the mouse β-globin promoter is preferred, with the CMV promoter being more preferred.

In the animal cells of the present invention, SNAT2 is overexpressed. Overexpression means that the expression of a gene exceeds the normal expression level in the host. By introducing a foreign gene encoding SNAT2 into a host and expressing the foreign gene in the host, an animal cell overexpressing SNAT2 can be obtained.

The expression level of SNAT2 in the animal cells of the present invention is preferably 3 times or more, more preferably 3.5 times or more, 4 times or more, as compared with the animal cells having no foreign gene encoding SNAT2. It is even more preferably fold or more, still more preferably 4.5 times or more, even more preferably 5 times or more, and particularly preferably 5.5 times or more. There is no upper limit, but it may be 30,000 times or less, or 10,000 times or less.

The expression level of SNAT2 can be examined by RT-PCR method (reverse transcription-polymerase chain reaction) or the like. The expression level of SNAT2 is preferably carried out by reverse transcription of mRNA and real-time PCR. The expression level of SNAT2 is preferably a relative expression level calculated by normalization. Normalization can be performed, for example, by comparative quantification using the expression level of housekeeping genes such as β-actin and HPRT1 as an endogenous control.

<Animal cells>
The cell in the present invention is not particularly limited as long as it is an animal cell. Animal cells include Chinese hamster ovary (CHO) cells, BHK cells, 293 cells, myeloma cells (NS0 cells, etc.), PerC6 cells, SP2 / 0 cells, hybridoma cells, COS cells, 3T3 cells, HeLa cells, Vero cells, etc. MDCK cells, PC12 cells, WI38 cells and the like can be mentioned. Among the above, CHO cells, BHK cells, 293 cells, myeloma cells (NS0 cells and the like), PerC6 cells, SP2 / 0 cells, hybridoma cells are particularly preferable, and CHO cells are further preferable. CHO cells are widely used in the production of recombinant proteins such as cytokines, coagulation factors, and antibodies. It is preferable to use CHO cells lacking dihydrofolate reductase (DHFR), and as the DHFR-deficient CHO cells, for example, CHO-DG44 can be used.

[Animal cell manufacturing method]
According to the present invention, the animal cell of the present invention comprises a step of introducing a gene encoding a target protein and a foreign gene encoding SNAT2 and linked to a promoter into the animal cell. A manufacturing method is provided.

It is preferable that the gene encoding the target protein and the foreign gene encoding SNAT2 are each incorporated into a vector and introduced into a host animal cell.

As a vector that can be used to introduce a gene into a host, for example, an expression vector derived from a mammal can be used, for example, pCMV6-Entry (manufactured by OriGene), pcDNA3 (manufactured by Invitrogen), pEGF-BOS. (Nucleic Acids. Res. 1990, 18 (17), p5322), pEF, pCDM8 (manufactured by Funakoshi), INPEP4 (manufactured by Biogen-IDEC) and the like, but are not particularly limited.

In addition, mRNA having poly A is known to be stable in cells. The gene encoding the target protein and the foreign gene encoding SNAT2 include poly A signals required for adding poly A to the gene, such as mouse β-globin poly A signal, bovine growth hormone poly A signal, and SV40 poly A signal. You may have.

The method for introducing the gene encoding the target protein and the foreign gene encoding SNAT2 into animal cells is not particularly limited, and can be carried out by a method known to those skilled in the art. For example, it can be carried out by electroporation, lipofection, calcium phosphate method, DEAE dextran method, a method using a cationic liposome DOTAP (manufactured by Roche Life Science Co., Ltd.), or a method using a viral vector. Among the above, electroporation is preferable.

When a gene is introduced into an animal cell, the gene is introduced into only a part of the cells subjected to the gene transfer, depending on the type of expression vector used and the gene transfer method. In order to identify and select the cell into which the gene has been introduced, for example, a gene encoding a selectable marker for resistance to an antibiotic may be introduced into the host cell together with the gene of interest. Preferred selectable markers include those that confer drug resistance, such as G418, hygromycin and methotrexate.

In the cells of the present invention, the gene encoding the target protein may be expressed in a transient expression system or a constitutive expression system, but may be expressed in a constitutive expression system. Is preferable.

In the cells of the present invention, the foreign gene encoding SNAT2 may be expressed in a transient expression system or a constitutive expression system, but may be expressed in a constitutive expression system. Is preferable.

The transient expression system is a method in which a cyclic plasmid is taken up into cells and expressed by a calcium phosphate method, an electroporation method, a lipofection method, or the like. The gene encoding the target protein and the foreign gene encoding SNAT2 are often present outside the chromosome.

In the constitutive expression system, a circular plasmid or a linear plasmid prepared by restriction enzyme treatment is incorporated into the cell by the calcium phosphate method, electroporation method, lipofection method, etc., and a part of the plasmid is inserted into the cell genome. This is a method for expressing the target protein. It is possible to maintain the expression of the gene encoding the target protein and the foreign gene encoding SNAT2 for a long period of time. Further, by introducing a drug resistance gene into a plasmid, drug selection becomes possible, and cells in which a gene encoding a target protein and a foreign gene encoding SNAT2 are maintained on a chromosome can be efficiently selected.

[Manufacturing method of target protein]
According to the present invention, there is provided a method for producing a target protein, which comprises culturing the animal cells of the present invention.

The target protein can be produced by culturing the animal cells of the present invention. Culturing can be carried out according to a known method.
As the medium used for culturing the animal cells of the present invention, the medium used for culturing normal animal cells can be used. For example, OptiCHO (Lifetechnologies, 12681011) medium, Dalveco modified Eagle's medium (DMEM), Eagle's minimum essential medium (MEM), RPMI-1640 medium, RPMI-1641 medium, F-12K medium, Ham F12 medium, modified Iscob medium. Dalbeco Medium (IMDM), McCoy 5A Medium, Leibovitz L-15 Medium, and EX-CELL ™ 300 Series (JRH Biosciences), CHO-S-SFMII (Invitagen), CHO-SF (Sigma-Aldrich) , CD-CHO (Invitrogen), IS CHO-V (Irvine Scientific), PF-ACF-CHO (Sigma-Aldrich) and the like can be used.

Serum such as fetal bovine serum (FCS) may be added to the medium, more preferably the culture medium may be cultured in a serum-free medium, and most preferably a completely synthetic medium.
The medium may be supplemented with additional components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, hydrolysates of plant proteins.

The pH of the medium varies depending on the cells to be cultured, but is generally pH 6.0 to 8.0, preferably pH 6.8 to 7.6, and more preferably pH 7.0 to 7.4.
The culture temperature is generally 30 ° C. to 40 ° C., preferably 32 ° C. to 37 ° C., more preferably 36 ° C. to 37 ° C., and the culture temperature may be changed during the culture.

The culture is _{preferably carried out in an atmosphere having a CO 2} concentration of 0 to 40%, preferably 2 to 10%.
The culturing time is not particularly limited, but is generally 12 hours to 90 days, preferably 24 hours to 60 days, and more preferably 24 hours to 30 days.
In culturing, medium exchange, aeration, and stirring can be added as needed.

The animal cells of the present invention can be cultured in a culture device (also referred to as a bioreactor) or in a suitable container other than that. The culturing equipment includes a fermenter tank culturing device, an air lift type culturing device, a culture flask type culturing device, a spinner flask type culturing device, a microcarrier type culturing device, a fluidized layer type culturing device, a holofiber type culturing device, and a roller bottle. It can be carried out using a type culture device, a filling tank type culture device, or the like.

The culture scale is generally 1 L to 20000 L, preferably 1 L to 10000 L, more preferably 200 L to 2000 L, and even more preferably 500 L to 2000 L.

As the culture, any method such as batch culture (batch culture), fed-batch culture (also referred to as fed-batch culture), or perfusion culture (perfusion culture) may be used, but fed batch culture or perfusion culture is preferable. ..

Batch culture is a discontinuous method in which cells are grown in a fixed volume of culture medium for a short period of time and then completely recovered. Cultures grown using the batch method experience an increase in cell density until maximum cell density is reached, after which they survive as media components are consumed and levels of metabolic by-products (such as lactate and ammonia) accumulate. Cell density decreases. Recovery is typically ^{(typically, 5~10 × 10 6 cells / mL} ) Maximum cell density performed when the has been achieved. The batch process is the simplest culture method, however, the density of viable cells is limited by nutrient availability, and once the cells reach maximum density, the culture is diminished and the production of the protein of interest is reduced. The duration of production of the protein of interest cannot be extended because waste product accumulation and nutrient depletion lead rapidly to culture decline (typically about 3-7 days).

Fed-batch culture is a culture method that improves the batch process by supplying bolus or continuous medium to replenish the consumed medium components. That is, in fed-batch culture, the culture form is suspension culture, which provides additional components to the culture at one or more time points after the start of the culture process. Additional components include nutritional supplements for cells that are depleted during the culture process and may include other supplements (eg, cell cycle inhibitory compounds).

Fed-batch cultures add additional nutrients throughout the culture period, which may result in higher cell densities and higher production of the protein of interest compared to batch cultures. Fed-batch cultures, unlike batch cultures, feed schedules and media to distinguish the period of cell proliferation (proliferative phase) to achieve the desired cell density from the period of discontinued or slow cell proliferation (production phase). Biphasic cultures can be made and sustained by manipulating the components. This may allow fed-batch cultures to achieve higher production of the protein of interest compared to batch cultures.

In fed batch culture, the seeded cell density of the cell culture is generally 0.2 × 10 ⁶ cells / mL or more and 1 × 10 ⁷ cells / mL or less, preferably 0.2 × 10 ⁶ cells / mL or more and 5 × 10 ⁶ cells / mL or less, more preferably 0.5 × 10 ⁶ cells / mL or more and 2.5 × 10 ⁶ cells / mL or less, still more preferably 0.5 × 10 ⁶ cells / mL or more 1 .5 × 10 ⁶ cells / mL or less.
In fed-batch culture, the viable cell rate during the culture period is preferably 60% or more and 100% or less, more preferably 70% or more and 100% or less, and further preferably 75% or more and 100% or less. ..

Fed-batch culture is a culture method in which fresh medium is added and used medium is removed at the same time, and there is a possibility that batch culture and fed batch culture can be further improved. Perfusion culture can be performed using, for example, an ATF (Alternating Tangential Flow Filtration) pump or a TFF (Tangential Flow Filtration) pump. According to perfusion culture, it is possible to achieve a high cell density of greater than 1 × ¹⁰ 8 cells / mL. A typical perfusion culture begins with a batch culture start-up that lasts for one or two days, after which fresh feed medium is added to the culture continuously, stepwise, and / or intermittently to remove used medium simultaneously. do. In perfusion cultures, methods such as sedimentation, centrifugation or filtration can be used to remove used medium while maintaining cell density. Perfusion culture can be performed using, for example, an ATF pump or a TFF pump.

The advantage of perfusion culture is that the culture in which the protein of interest is produced is maintained for a longer period of time than batch culture or fed-batch culture. However, in order to maintain long-term perfusion cultures, especially perfusion cultures at high cell densities, medium preparation, use, storage and disposal are required. Fed-batch culture requires a large amount of nutrients, and the production cost of the target protein tends to be higher than that of batch culture and fed-batch culture. In addition, by selecting the membrane pore size, it is possible to continue the culture while recovering the antibody from the system. Therefore, the retention time of the antibody in the culture solution is shortened and the chemical change is reduced, so that the quality of the antibody is reduced. Can be kept high.

It is also possible to carry out a culture in which a fed batch culture and a perfusion culture are combined. As an example, fed-batch cultures with a bolus feed can be used to maintain the culture of cells in the proliferative phase, and then perfusion cultures can be used for the production of the protein of interest.

Perfusion may be in any form of continuous, gradual, intermittent or a combination thereof. Animal cells are retained in the culture and removed. The used medium may be substantially free of cells or may have much less cells than the culture. The target protein expressed by cell culture can be retained or recovered in the culture by selecting the membrane pore size. The cell density may be reduced (cell bleeding) by extracting a part of the culture solution together with the cells and adding the same amount of fresh medium so that the cell density during the culture does not become excessive.

In perfusion culture, the seeded cell density of the cell culture is generally 0.2 × 10 ⁶ cells / mL or more and 3 × 10 ⁷ cells / mL or less, preferably 0.2 × 10 ⁶ cells / mL or more 1 It is × 10 ⁷ cells / mL or less, and more preferably 0.5 × 10 ⁶ cells / mL or more and 1 × 10 ⁷ cells / mL or less.
In perfusion culture, the viable cell rate during the culture period is preferably 80% or more, more preferably 85% or more, still more preferably 90% or more over the entire period.

In perfusion culture, the maximum cell density is preferably 2 × 10 ⁸ cells / mL or less, more preferably 1.5 × 10 ⁸ cells / mL or less, and even more preferably 1.0 × 10 ⁸ cells / mL. It is less than mL.

The perfusion ratio in the perfusion culture is preferably 0.3 vvd or more and 5.0 vvd or less, and more preferably 0.3 vvd or more and 1.5 vvd or less. However, vvd represents the following.
vvd = (volume of fresh medium / working volume of reactor / day, daily supply culture solution volume / culture solution volume)

The seeded cell density in the cell culture and the maximum cell density reached in the culture can be determined by measuring the number of cells by a conventional method and dividing the number of cells by the amount of the culture medium.
The viable cell rate (survival rate) during the culture period is obtained by dividing the number of viable cells by (the number of viable cells + the number of dead cells). For example, the number of cells can be measured using Vi-CELL XR (Beckman Coulter).

The target protein produced by the above culture can be purified. For the separation and purification of the target protein, the separation and purification method used for ordinary proteins may be used. For example, by appropriately selecting and combining a chromatography column such as affinity chromatography, a filter, ultrafiltration, salting out, dialysis, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, isoelectric focusing, etc., the target protein. Can be separated and purified, but is not limited thereto. The concentration of the target protein obtained above can be measured by absorbance measurement, enzyme-linked immunosorbent assay (ELISA) or the like.

Examples of the column used for affinity chromatography include a protein A column and a protein G column. Chromatography other than affinity chromatography includes, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography and the like. These chromatographies can be performed using liquid phase chromatography such as HPLC (high performance liquid chromatography) or FPLC (fast performance liquid chromatography).

The target protein can be modified or the peptide can be partially removed by allowing an appropriate polypeptide modifying enzyme to act before or after purification. As the polypeptide modifying enzyme, for example, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, glucosidase and the like are used.

[Use of target protein]
When the target protein produced by the method of the present invention has biological activity useful as a pharmaceutical product, the target protein is formulated by mixing it with a pharmaceutically acceptable carrier or additive. Can manufacture pharmaceutical products.

Examples of pharmaceutically acceptable carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water. Sex Dextran, Sodium Carboxymethyl Starch, Pectin, Methyl Cellulose, Ethyl Cellulose, Xanthan Gum, Rubber Arabia, Casein, Agar, Polyethylene Glycol, Diglycerin, Glycerin, Propylene Glycol, Vaseline, Paraffin, Stearyl Alcohol, Stealic Acid, Human Serum Albumin (HSA) , Mannitol, sorbitol, lactose, pharmaceutically acceptable surfactants and the like.

For example, when used as an injectable preparation, the purified target protein is dissolved in a solvent such as physiological saline, a buffer solution, a glucose solution or the like, and an adsorption inhibitor such as Tween80, Tween20, gelatin, human serum albumin or the like is used. Can be used. Alternatively, the target protein may be freeze-dried in order to form a dosage form that dissolves and reconstitutes before use, and examples of the excipient for freeze-drying include sugar alcohols such as mannitol and glucose. Sugars can be used.

The method for administering the target protein may be either oral administration or parenteral administration, but parenteral administration is preferable. For example, injection (for example, systemic or local administration by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.), nasal administration, pulmonary administration, transdermal administration, and the like can be mentioned.

The dose of the target protein is appropriately selected depending on the type of the target protein, the type of disease to be treated or prevented, the age of the patient, the severity of the disease, and the like. Generally, it is in the range of 0.001 mg to 1000 mg per 1 kg of body weight at a time, but is not particularly limited.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.

<Example 1> Preparation of animal cells A vector encoding the human SNAT2 gene (OriGene, Cat # RC2018892) was purchased, and the expression cassette (promoter, open reading frame, terminator) of the SNAT2 gene was used as primer 1 (SEQ ID NO: 3). And primer 2 (SEQ ID NO: 4) was used to amplify by polymerase chain reaction (PCR).

Antibody 1 (denosumab, https: //www.drugbank.ca/drugs/DB06643), antibody 2 (brosozumab, https: //www.drugbank.ca/drugs/DB12560) or antibody 3 (anti-MUC1 antibody, Table 20). An amplified SNAT2 expression cassette was inserted into a vector that co-expresses the L chain and H chain of each antibody (No. -517691). The construction of the L-chain and H-chain co-expression vector and the introduction into cells were carried out according to Example 2 of Japanese Patent Application Laid-Open No. 2016-517691.

All host CHO-DG44 cells prior to the introduction of the antibody and SNAT2 gene were cultured in an incubator at _{37 ° C. in a 5% CO 2 atmosphere.} 20 mL containing HT Supplement (x100) (Lifetechnologies, 11067-030) obtained by introducing the above vector into host cells 5 × 10 ^{6 cells using an electroporation method (Lonza, 4D-Nuclefector) and diluting 100-fold.} After suspension in OptiCHO (Life technologies, 12681011) medium, the cells were seeded in a T75 flask. One day later, the medium was replaced with OptiCHO (Life technologies, 12681011) medium containing 175 nmol / L methotrexate (MTX) (Wako Pure Chemical Industries, Ltd.) without HT supplements. Microscopic observation of the T75 flask was started 3 weeks after the gene transfer, the culture volume was gradually expanded to 20 mL from the flask in which cell proliferation was observed, seeded in a 125 mL shaking flask (Corning), and shaken at 140 rpm. It was cultured.

By the above method, antibody 1-SNAT2 cells, antibody2-SNAT2 cells, and antibody 3-SNAT2 cells that forcibly express the human SNAT2 gene and antibody 1 or antibody 2 or antibody 3 were established. In addition, gene transfer was carried out in the same manner using a vector expressing antibody L chain and H chain to construct antibody 1 cell, antibody 2 cell, and antibody 3 cell in the control group.

Primer-1: CCGCGGTCATAGCTGTTTCCTGAAC (SEQ ID NO: 3)
Primer-2: CAGCTATGACCGCGGTTAATGGCCACCTCCAGGTGCATTGTGT (SEQ ID NO: 4)

<Example 2> Fed-batch culture experiment A fed-batch culture experiment was carried out using 8 levels each of antibody 1-SNAT 2 cells, antibody 2-SNAT 2 cells, antibody 3-SNAT 2 cells and antibody 1 cell, antibody 2 cells, and antibody 3 cells. ..
The cells were suspended in 15 mL of OptiCHO (Lifetechnologies, 12681011) medium at a cell density of ^{5 × 10 5 cells / mL and automatically cultured with an AMBR15 incubator (Sartorius Stedim).} From the 2nd day to the 13th day of the start of the culture, feed medium (Cellboss7a, 7b, GE healthcare) was added daily at a ratio of 2% to the initial culture volume. Sampling was performed every 3-4 days to measure cell density, culture components and antibody concentration over time. On the 14th day after the start of culturing, the culture broth was collected, and cells and cell debris were removed using a 0.22 μm depth filter (Merck Millipore). When the antibody concentration in the supernatant and the antibody productivity (Qp) per cell were measured by liquid chromatography, the antibody concentration increased by 121% for antibody 1, 25% for antibody 2, and 56% for antibody 3 (FIG. 1). And Table 1). In addition, Qp increased by 127% for antibody 1, 34% for antibody 2, and 74% for antibody 3 (FIGS. 2 and 2). It is considered that the antibody productivity was improved because the protein biosynthesis was activated by the expression of SNAT2. On the other hand, as a result of measuring the cell diameter with Vi-CELL (Beckman Coulter), surprisingly, the size of each cell was antibody 1-SNAT2 cells (average 15.8 μm) and antibody 2-SNAT2 cells (15.8 μm on average). It was found that the average was 16.3 μm) and the antibody 3-SNAT2 cells (average 16.5 μm), and the control group had 1 antibody cell (average 16.3 μm), 2 antibody cells (average 15.8 μm), and antibody 3. Compared with the cells (average 16.2 μm), the cell diameter did not necessarily increase and showed almost the same level (FIGS. 3 and 3). In addition, the cumulative number of viable cells (IVCD) during the culture period, which indicates cell proliferation, was about the same or slightly higher than that of the control group (FIGS. 4 and 4). In FIGS. 1 to 4, the left shows only the antibody, and the right shows the antibody-SNAT2. This proved that the expression of SNAT2 did not promote the differentiation of CHO cells, and maintained the cell proliferation ability necessary for the establishment of antibody-producing cells and the production of antibodies.
The integrated viable cell density (IVCD) was measured by the following method. On days 0, 3, 7, 10 and 14, the viable cell density (VCD) is measured by Vi-CELL XR (Beckman Coulter) and the respective values are V0, V3, V7, V10 and V14. The integrated viable cell density (IVCD) is calculated by the following formula.
Integral viable cell density (IVCD) = {(V0 + V3) x (3-0) / 2} + {(V3 + V7) x (7-3) / 2} + {(V10 + V7) x (10-7) / 2} + {(V14 + V10) x (14-10) / 2}

The antibody productivity Qp [pg / cell / day] per cell was calculated by the following formula.

Pti [g / L] = concentration of purified product on culture day ti Xti [cell / day] = viable cell density on culture day ti

The approximate value of the integral was calculated by obtaining the area under the growth curve from time t1 to t2 as the area of the trapezoid.

Claims (14)

An animal cell having a gene encoding a target protein and a foreign gene encoding SNAT2 and linked to a promoter, and overexpressing SNAT2. The animal cell according to claim 1, wherein overexpression is constitutive. The animal cell according to claim 1 or 2, wherein the foreign gene encoding SNAT2 has a base sequence having 90% or more sequence identity with the base sequence set forth in SEQ ID NO: 1. The animal cell according to any one of claims 1 to 3, wherein the foreign gene encoding SNAT2 contains the nucleotide sequence set forth in SEQ ID NO: 1. The animal cell according to any one of claims 1 to 4, wherein the animal cell is a CHO cell. The invention according to any one of claims 1 to 5, further comprising a step of introducing a gene encoding a target protein and a foreign gene encoding SNAT2 and linked to a promoter into an animal cell. How to make animal cells. The method according to claim 6, wherein the step of introducing a foreign gene encoding SNAT2 and linked to a promoter is performed by electroporation. A method for producing a target protein, which comprises culturing the animal cell according to any one of claims 1 to 5. The method according to claim 8, wherein the culture is fed-batch culture. The method according to claim 9, wherein the seeded cell density of the cell culture is 0.2 × 10 ⁶ cells / mL or more and 5 × 10 ^{6 cells / mL or less.} The method according to claim 10, wherein the viable cell rate during the culture period is 60% or more over the entire period. The method according to claim 8, wherein the culture is perfusion culture. The method according to claim 12, wherein the seeded cell density of the cell culture is 0.2 × 10 ⁶ cells / mL or more and 1 × 10 ^{7 cells / mL or less.} 13. The method of claim 13, wherein the viable cell rate during the culture period is 90% or more over the entire period.

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